Micro-fluidic system using micro-apertures for high throughput detection of cells

ABSTRACT

A microfluidic detection system for micrometer-sized entities, such as biological cells, includes a detector component incorporating a plate with a plurality of opening, the plate separating two chambers, one in communication with a fluid source containing target cells bound to magnetic beads. The openings are sized to always permit passage of the magnetic beads therethrough into a lower one of the chambers and are further sized to always prevent passage of the target cells from the upper one of the chambers. The detector component further includes a magnet positioned to pull unbound magnetic beads through the openings and to capture target cells bound to magnetic beads on the surface of the plate. The microfluidic detection system includes a pump flowing the fluid through the detector component at high flow rates of milliliters per minute for high throughput detection of target cells.

PRIORITY CLAIM

The present application is a continuation of Ser. No. 14/001,963, filedon Oct. 8, 2013, which is a national stage under 35 U.S.C. § 371 ofinternational application PCT/US2012/032356, filed on Apr. 5, 2012,which claims priority to provisional application Ser. No. 61/471,762,filed on Apr. 5, 2011, in the name of the same inventors, the entiredisclosure of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention generally relates to microfluidics andparticularly to detecting targeted cells present in sample fluids.

BACKGROUND

Circulating tumor cells (or CTCs) are rare cells present in the blood ofmetastatic cancer patients. Quantitative detection of CTCs is importantfor early detection of cancer as well as monitoring of the diseaseprogression and response to therapy. CTC count correlates with overalltumor burden and can often serve as more reliable indicators ofmetastatic disease than molecular disease markers. For example, thelevel of prostate specific antigen (PSA) can often rise due to benignprostate hyperplasia (common in people over 60) and hence may notnecessarily indicate cancer.

The presence of a significant number of CTCs can be a reliable indicatorof the presence of cancer. Also, possible recurrence after surgery canbe detected much earlier by CTCs than by most molecular markers. Anotheradvantage is that CTCs can be further interrogated after detection;sequencing of the genome and transcriptome could reveal the mutationsthat had led to cancer as well as the expression levels of the genes inquestion. The cells can also be cultured, grown and tested withdifferent combinations of chemotherapeutic agents for drug discovery andpersonalized medicine.

Detecting CTCs however is a challenging task because of their scarcityin blood samples, as few as single cell in multiple milliliter (mL)blood samples. The current favored approach to detecting whole cells inclinical and laboratory settings is flow cytometry, wherein labeledcells are detected as they flow in single file through an opticaldetector. This technology is used widely from vaccine analysis tomonitoring of AIDS. However, the high cost and large size of flowcytometers usually limits this testing approach to central facilitiesshared by many users. Furthermore, since the cells have to pass througha sensing portion of the flow cytometers in a single file manner,volumetric sample throughput is relatively low and cytometers need torun for long times to analyze large samples. For so-called “rare”cells—i.e., cells that are scarce in a fluid sample, such as CTCcells—relatively large volume samples may be required to find the cells.In this instance, the current flow cytometers can be prohibitivelyexpensive for frequent diagnostic usage.

Microfluidic cell detectors have been developed to overcome the cost andsize limitations of traditional flow cytometers in certain applications.These sophisticated systems can successfully interrogate small samples,on the order of μLs (microliters), but such systems have been found tohave limited capability for analyzing large samples, on the order ofmultiple mLs. Most microfluidic systems offer good performance inanalyzing small, microliter- or nanoliter-sized sample volumes. However,because of their micrometer dimensions, microfluidic “lab-on-a-chip”detectors need many hours to process large, milliliter-sized samplevolumes. Slow flow rates in microfluidic assays are usually aconsequence of the microscale dimensions of the sensing channels. Thesedimensions are necessary to increase the probability of a rare cell(i.e., a CTC) binding on the walls of the microchannels and in somecases to increase the signal-to-noise ratio of the underlying detectionmechanism. Thus, the prior microfluidic cell detectors can be generallyinefficient and can require prohibitively long analysis times foranalyzing the large volume samples necessary for the detection of raretargets like CTCs.

In one microfluidic detection system developed by the Toner and Habergroups of Massachusetts General Hospital, a lab-on-a-chip is populatedwith antibody-functionalized 100 μm diameter posts spaced 50 μm apart tocreate fluid flow paths. In another chip design, the posts were replacedwith a herringbone structure to actively assist mixing of the cells andincrease their probability to bind the functionalized walls. In thesestudies, flow rates used with clinical samples were on the order of 1 mLper hour, at which rate processing a typical 7.5 mL blood sample couldtake many hours. In order to reduce the fluidic transport times to amanageable level of minutes rather than hours, the flow rate throughthese prior systems would have to be increased by one or two orders ofmagnitude. In general the necessary modifications to prior microfluidicsystems can be problematic because: 1) the fluidic resistance of themicron-sized flow channels and the associated macro-to-micro connectionswould be very high; 2) a high flow rate through a small cross-sectionalarea would result in a high “linear speed” which would create shearstresses beyond levels that could be sustained by the antibody/cellbinding on the device wall and lead to detachment of the cells; and 3)too high a linear speed would detrimentally affect the captureefficiency of the target cells in the first place. Increasing the sizeof the channels would allow higher flow rates but this wouldsignificantly reduce the probability of the target cells' interactionwith the functionalized walls.

In the case of other microfluidic devices that use electronic detectiontechniques, larger dimensions would reduce the device's detectionsensitivity since most microdevices need some form of focusing oftargets onto a small sensor area for detection. Other researchers haveparallelized their microfluidic detectors (many micro-channels side byside) to overcome the throughput problem. However, the flow rates thatare used can be on the order of only 10 microliters (μLs) per minute,which can lead to hours of time to process the large volume samplesnecessary for CTC detection.

A high-throughput yet relatively simple and robust rare cell detectionsystem would be highly beneficial in many research and clinicalsettings. Therefore, a sensor apparatus is needed that can detect rarecells, such as CTCs, in whole blood in a high-throughput manner by whichsample fluids at rates of milliliters per minute (as opposed tomicroliters per minute) are processed to capture the contained cells.Such a system would also be highly useful in detecting various othertypes of cells, bacteria and spores present in sample fluids or in theenvironment.

SUMMARY

According to one aspect of the current teachings a fluidic systemincluding a detector component with micro-apertures is disclosed whichis configured to detect target cells bound by recognition elements, suchas magnetic beads, in a high-throughput analysis for flow rates ofmilliliters per minute. In particular, a microfluidic detection systemis provided for detection of target cells in a fluid containing aquantity of magnetic beads and a quantity of target cells bound to oneor more magnetic beads, in which each target cell bound to a magneticbead has a smallest effective dimension greater than a smallesteffective dimension of each magnetic bead. In one aspect, the systemcomprises a detector component including a body defining a reservoir anda sensor chip in the form of an apertured plate disposed within thereservoir and separating the reservoir into a first chamber and a secondchamber. The plate includes a plurality of micro-openings, each openinghaving smallest effective dimension greater than the smallest effectivedimension of each magnetic bead and less than the smallest effectivedimension of each target cell bound to a magnetic bead. In one aspect,the first chamber of the reservoir has a first inlet and a first outlet,in which the first inlet is fluidly connectable to a source of the fluidcontaining the target cells, while the first outlet is fluidlyconnectable to a collection vessel.

The detector component further includes a magnet disposed relative tothe reservoir so that the second chamber of the reservoir and theapertured plate are situated between the magnet and the first chamber ofthe reservoir. The magnet configured to generate a magnetic forcesufficient to attract magnetic beads in the first chamber of thereservoir to the second chamber of the reservoir. The microfluidicdetection system further comprises a pump for continuously flowing thefluid from the source of the fluid containing the target cells throughthe first chamber of the reservoir.

A method is provided for detecting target cells in a fluid containing aquantity of magnetic beads and a quantity of target cells bound to oneor more magnetic beads, in which each target cell bound to a magneticbead has a smallest effective dimension greater than largest effectivedimension of each magnetic bead. In one aspect, the method comprises:continuously flowing the fluid through a first chamber of a reservoirseparated from a second chamber of the reservoir by an apertured plate,each opening in the plate having a smallest effective dimension greaterthan the smallest effective dimension of each magnetic bead and lessthan the smallest effective dimension of each target cell bound to amagnetic bead; and applying a magnetic force beneath the apertured platesufficient to draw magnetic beads not bound to a target cell through theapertures into the second chamber and sufficient to hold the targetcells bound to one or more magnetic beads against the surface of theapertured plate within the first chamber of the reservoir.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a schematic view of a microfluidic detection system accordingto the present disclosure.

FIG. 1B is a schematic view of a portion of the system shown in FIG. 1Bmodified in accordance with an alternative embodiment.

FIG. 2 is a side cross-sectional view of a detector component accordingto the present disclosure for use in the detection system shown in FIG.1.

FIG. 3 is a plan view of a sensor chip micro-perforated plate for use inthe system shown in FIG. 1A.

FIG. 4 is a side representation of the operation of the sensor chipshown in FIG. 44 to capture target cells and extract unbound magneticrecognition elements.

FIGS. 5A-D are schematic views of the microfluidic detection system ofFIG. 1A, showing fluid flow paths in different stages of operation ofthe system.

FIG. 6 is a bright-field microscopic picture of target cells captured ona sensor chip in accordance with a disclosed embodiment.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and described in the following written specification. It isunderstood that no limitation to the scope of the invention is therebyintended. It is further understood that the present invention includesany alterations and modifications to the illustrated embodiments andincludes further applications of the principles of the invention aswould normally occur to one of ordinary skill in the art to which thisinvention pertains.

The present disclosure provides a micro-fluidic system with a detectorcomponent having an apertured plate or chip configured to providehigh-throughput analysis of mLs (milliliters) per minute of a fluidsample flowing through relatively large (millimeter (mm) as opposed tomicrometer (μm)) flow channels. The fluid sample may be a bodily fluid,including but not limited to whole blood, processed blood, serum,plasma, saliva and urine, or environmental fluids, including but notlimited to fluid samples from rivers, sewage lines, water processingfacilities and factories. Using functionalized beads capable of bindingto target cells in three dimensions, the system eliminates the need forchemical affinity-based binding of the cells to a stationarytwo-dimensional chip surface. The system uses both convective fluid flowto assist in mass transport of target cells bound by functionalizedmagnetic beads, and magnetic sieving of the bound cells onto a plate orchip with micro-apertures which captures the cells but allows freemagnetic beads (i.e., those not bound to target cells) to pass throughthe apertures. In certain embodiments the magnetic beads simultaneouslyserve to: 1) affinity-based bind specific target cells; 2) magneticallytransport the bound cells to the plate or chip surface; and 3) generaterecognition or labeling signals for detection. The novel fluidic systemof the present disclosure can analyze large amounts of sample fluids,including clinically significant amounts of bodily fluids such as wholeblood, in a relatively short amount of time.

FIG. 1A depicts a schematic of a micro-fluidic system 10 according toone embodiment of the present disclosure. The system 10 includes adetector component 12 having a first flow segment 13 and a parallelsecond flow segment 14. The first flow segment 13 is provided with aninlet 13 a and an outlet 13 b, in which the inlet is connected by aninlet conduit 35 to a sample fluid source S. The outlet 13 b isconnected by outlet conduit 36 to a collection vessel CC, which issuitable for collecting and storing target cells isolated by themicrofluidic detection system 10. The second inlet 14 a is connected byan inlet conduit 38 to a source of a buffered or physiologically inertor non-reactive solution B. The second outlet 14 b is connected by anoutlet conduit 39 to a collection vessel BC for collecting bufferedsolution exiting the detector component 12.

In one embodiment a pump 40 is provided for flowing fluid from thesample source S through the first flow segment 13 of the detectorcomponent 12. In the embodiment shown in FIG. 1 the pump 40 isintegrated into the outlet conduit 36 to draw fluid through the detectorcomponent. However, it is contemplated that the pump 40 may be situatedwithin the inlet conduit 35 as desired. The embodiment of FIG. 1Afurther includes a second pump 41 that is integrated into the outletconduit 14 b to flow the buffer fluid from source B through the secondflow segment 14 of the detector component 12. The pump 41 may beprovided in the inlet conduit 38 to draw buffer fluid from the source Band pump it through the second flow segment 14. Alternatively the samepump, such as pump 40, may be used to pump both the sample fluid fromthe source S through the first flow segment 13 and the fluid from sourceB through the second flow segment 14, as depicted in the enlargedsegment shown in FIG. 1B. In this embodiment the two outlet conduits 36,39 are connected to the pump 40 through a bi-directional valve V6 thatis controllable to selectably connect one or the other outlet for flowthrough the pump, according to a fluid flow protocol discussed herein.

Returning to FIG. 1A, the microfluidic system 10 incorporates bypasslines that are selectively activated according to a fluid flow protocoldescribed below. A bypass line 42 is provided between the buffer sourceB and the inlet conduit 35 to the first flow segment 13 of the detectorcomponent. A valve V1 is operable to control flow of buffer solutionfrom the source B into both inlet conduits 35, 38, while a valve V2 isoperable to control flow of buffer solution into the first inlet conduit35.

In a second bypass path, a bypass line 43 is connected between the firstoutlet conduit 36 and the first inlet conduit 35. This bypass line thusreturns fluid exiting the first flow segment 13 of the detectorcomponent back to the inlet 13 a for the first flow segment. A valve V3controls fluid flow through the second bypass line 43. A third bypasspath includes the bypass line 44 from the second outlet conduit 39 tothe source S containing the sample fluid. A valve V4 is configured todirect the flow of buffer fluid exiting the second flow segment 14either to the buffer collection vessel BC or to the third bypass line44. The system 10 is provided with a control module 45 that is operableto control the pump(s) 40 (and 41 if present) as well as the valves tocontrol the flow of fluids through each flow segment 13, 14 of thedetector component 12, according to a flow protocol described herein.

The microfluidic system 10 may be configured to accept sample fluidsfrom multiple sources S_(i). The multiple sources S_(i) may be connectedin series or in parallel, with appropriate valving to connect theparticular source to the inlet conduit 35 to the first flow segment 13of the detector component. The system may be further modified to includeadditional detector components 12 i connected to the outlet conduit 36from the first flow segment 13, by way of a control valve V5.

The flow of sample fluid and buffer solution through the system 10, andparticularly through the detector component 12, has thus far beendescribed. Details of the detector component 12 and its function areillustrated in FIGS. 2-4. Turning first to the cross-sectional view inFIG. 2, the detector component 12 includes a body 15 that may be in theform of two halves 15 a, 15 b that are combined to form the completecomponent. The body 15 defines a reservoir 16 that is separated into afirst chamber 16 a and a second chamber 16 b by a sensor chip in theform of a micro-apertured plate 18. The first chamber 16 a is incommunication with the first flow inlet 13 a and the first flow outlet13 b and thus forms the first flow segment 13 of the detector component.A first inlet channel 22 communicates between the first chamber 16 a andthe inlet 13 a, while a first outlet channel 23 communicates with theoutlet 13 b. In the illustrated embodiment, the two channels are angledtoward the reservoir 16, or defined at a non-planar angle relative tothe reservoir, so that the target cells and sample fluid do notaccumulate within the channel 22 or inlet conduit 35.

The second chamber 16 b is in communication with the respective secondinlet and outlet 14 a, 14 b to form the second flow segment 14 of thedetector component. An inlet channel 25 communicates between the secondchamber and the second inlet 14 a, which an outlet channel 26communicates with the second outlet 14 b. The two channels 25, 26 can beangled but need not be since the second chamber 16 b is connected to thebuffer solution source B and no target cells flow through this secondfluid flow path 14.

The reservoir 16 may be open at one side of the detector component 12,with the reservoir opening sealed and closed by a window or viewingpanel 20. The viewing panel 20 is oriented to provide an unobstructedview of the apertured plate 18 within the reservoir 16. In oneembodiment the viewing panel 20 is optically transparent to permitdirect visualization of the surface of the apertured plate.

The sensor chip or apertured plate 18 may be supported by a platemounting 28, formed around the perimeter of the plate, that is trappedbetween the two body halves 15 a, 15 b when they are coupled together. Aseal 29 may be provided on one or both sides of the plate mounting 28 toensure a fluid-tight seal between the two chambers 16 a, 16 b. Detailsof the apertured plate are shown in FIGS. 3-4. In particular, the plate18 includes an upper surface 51 and a plurality of micro-sized aperturesor openings 50 defined therethrough. In one embodiment the openings aregenerally uniformly sized and have a largest effective dimension d thatis less than the smallest effective dimension of a target cell T (FIG.4) that is to be detected. Moreover, the smallest effective dimension dof the plate openings is greater than a largest effective dimension ofmagnetic recognition elements M. For the purposes of the presentdisclosure, the term “effective dimension” refers to a dimension of aparticular element measured along a particular axis. For a circularopening in the plate or a spherical magnetic bead, the smallest andlargest effective dimensions are the same and are simply the diameter ofthe opening or bead. For an oblong opening, the largest effectivedimension is the length of the opening along its long axis, while thesmallest effective dimension is the width along the short axis. Thetarget cells may not exhibit a uniform three-dimensional shape, (such asa sphere) so the cell will have a different dimension depending upon theaxis of measurement. For cells of this type, the term “smallesteffective dimension” refers to the smallest of those measurements. Thus,the relative effective dimensions of the plate openings are such that amagnetic bead can always pass through any opening no matter how the beadis oriented, while a target cell can never pass through any openingregardless of how it is oriented.

The microfluidic system 10 is configured to detect and isolate targetcells T that are bound to recognition elements M. Thus, the fluid sourceS holds a sample fluid that contains target cells, for instance a bloodsample of a patient that contains circulating tumor cells (CTCs) or asample that has exemplary tumor cell lines such as lymph node carcinomaof the prostate cells (LNCaP) or ovarian cancer cells (IGROV). The fluidsample further contains recognition elements in the form of magneticbeads M that bind to the target cells. Details of the target cells andrecognition elements will follow, but with respect to the openings 50 inplate 18 it can be appreciated that the size of the openings iscalibrated so that any free magnetic beads M (i.e., beads that have notbound to a target cell T) will pass freely through the opening, such asthe beads M_(X) on the right side of the plate in FIG. 4. On the otherhand, the openings 50 are sized so that the target cells T cannot passtherethrough, with or without any magnetic beads bound thereto, such asbeads M_(B).

The significance of the magnetic beads M can be appreciated by referringback to FIG. 2. In particular, the body 15 of the detector component 12includes a magnet 32 mounted within a cavity 31 beneath the secondchamber 16 b. In particular, the magnet 32 is positioned so that themagnetic force attracts magnetic beads M within the first chamber 16 atoward the apertured plate 18. It is this magnetic force that pulls thefree magnetic beads M_(X) through the openings 50, as illustrated inFIG. 4, even while the beads are under the influence of a fluid flow Fthat is substantially parallel to the surface 51 of the plate 18. Thissame magnetic force also attracts beads M_(B) that are bound to a targetcell T, which are also under the influence of the parallel fluid flow F.However, since the target cell T is too large to pass through anyopening the magnetic force serves to hold the bound target cell againstthe surface 51 of the apertured plate 18. In certain instances thetarget cell T is large enough relative to the magnetic beads M to haveseveral beads M_(B) bound to the cell. As depicted in FIG. 4, some ofthe bound beads M_(B) extend partially into an opening 50. The beadsM_(B) are held within the opening by the magnetic force, which not onlyholds the bound target cell T to the plate surface 51 but also restrainsor “locks” the cell against translating along the surface or beingwashed away under the influence of the fluid flow F. Thus, the boundbeads M_(B) not only capture target cells but also help prevent thecaptured target cells from bunching up or collecting at the outlet endof the first chamber 16 a.

The magnet 32 is calibrated relative to the magnetic beads M to exert amagnetic force sufficient to pull the beads toward the apertured platebut not so strong as to break the bound beads M_(B) away from a targetcell T captured on the plate surface 51. The magnetic force is alsosufficiently strong to pull the beads and target cells out of the fluidflow F that tends to propel the beads and cells in a flow path parallelto the surface 51 of the sensor chip plate 18. In a specific example themagnet is a NdFeB Cube Magnet (about 5×5×5 mm) with a measured fluxdensity and gradient of 0.4 T and 100 T/m, respectively. Other magnetsare envisioned including but not limited to larger or smaller permanentmagnets made of various materials, and electromagnets that arecommercially available or manufactured using standard ormicrofabrication procedures and that are capable of generatingtime-varying magnetic fields. In the illustrated embodiment of FIG. 2the magnet 32 is housed within a cavity 31 formed in the bottom half 15b of the housing. However, the magnet may be affixed to or supportedrelative to the outside of the detector component 12 provided that it isoriented in a manner to draw the magnetic beads M from the first chamber16 a to the second chamber 16 b. It is further contemplated that themagnet 32 may be associated with the detector body 15 so that thedistance of the magnet from the apertured plate 18 may be varied tothereby vary the magnetic force applied to the magnetic beads in thefirst chamber 16 a. The magnetic force may thus be calibrated to aparticular magnetic bead. In addition, the magnet 32 may be moved toremove the magnetic force entirely according to a flow protocol for themicrofluidic system 10. Removal of the magnetic field can facilitate theremoval of captured target cells from the plate surface so that thetarget cells may be transported or flushed to a separate collectionvessel CC.

In another embodiment, a magnetic field may be applied from the top ofthe detector component 12 or directly above the surface 51 of the sensorchip plate 18. This magnetic field may thus “levitate” the capturedtarget cells off the surface 51 to further facilitate their removal. Itis contemplated in this embodiment that the magnetic field of the magnet32 is disrupted as described above so that the magnetic field appliedfrom the top of the component does not “compete” with the originalcapturing magnetic field.

As explained above, the pump(s) 40 (41) and the valves V1-V5 arecontrolled according to a flow protocol adapted to; a) prepare thedetector component 12 to receive a fluid containing bound target cells;b) capture the bound target cells; c) flush unbound magnetic beads; andd) extract captured target cells. In a first step of the protocol, thesystem is primed with an non-reactive or buffered solution from sourceB. The solution from source B is preferably non-reactive to the targetcells T, to the recognition elements or magnetic beads B, and to anyligands, antibodies, aptamers, peptides, low molecular weight ligands,or antigens used to functionalize and bind the recognition elements. Ina specific embodiment the solution may be a phosphate buffered saline(PBS). Referring to FIGS. 1A and 5A, the reservoir 16 is initiallyflooded with PBS by opening valve V1, moving valve V4 to close thebypass line 44 but open the flow path to the collection container BC,and moving valve V2 to close the inlet conduit 35 to the sample source Sand open the conduit to the bypass line 42. Valves V3 and V5 are closedso that all of the fluid exiting the detector component 12 is fed to thebuffer collection container BC. The buffered solution PBS flows freelythrough both chambers 16 a, 16 b and through the apertured plate 18 sothat all fluid flows through the second outlet channel 26 and secondoutlet 14 b into outlet conduit 39 and collection container BC. Incertain embodiments it may be desirable to open valve V5 to pump PBSfrom chamber 16 a into collection vessel CC in order to avoid anypressure increase within the chamber. The pump 41 is thus activated tocontrol the flow of PBS through both flow segments 13, 14. In thealternative configuration of FIG. 1B, the pump 40 provides the motiveforce for fluid flow with the valve V6 open to both outlets 13 b, 14 bbut with pump discharge to only the second outlet conduit 39. It can beappreciated that this initial flow of PBS through the system will purgethe air from the reservoir and channels.

With the detector component primed, the buffered solution fluid circuitis deactivated by closing the valves V1 and V4, closing the bypass line42 at valve V2, and deactivating pump 41. The first chamber 16 a is nowready to receive the sample fluid from source S by opening valve V2 tothe inlet conduit 35 and valve V5 to the collection vessel CC, asillustrated in FIG. 5B. Pump 40 is activated to draw the sample fluidfrom the source S through the detector component 12, and moreparticularly to pull the sample fluid through the first inlet 13 a intothe first chamber 16 a. The magnet 32 is activated to pull the magneticbeads M to the apertured plate 18, as depicted in FIG. 4. It can beappreciated that the rate of flow F of the sample fluid is calibrated sothat the fluid pressure does not overcome the magnetic force. By way ofnon-limiting example, the pump 40 may be configured to produce a flowrate of several mLs/min, which is significantly faster than the mLs/hourrates of prior microfluidic systems. In one specific embodiment theinlet and outlet channels 22, 23 may have a smallest effective dimensionof 0.5 mm so that a 1 mL/min flow rate may generate a linear flowvelocity of about 3 mm/sec in the channels and about 0.7 mm/sec throughthe reservoir 16. These linear velocities are nearly 100 fold lower thanvelocities believed to cause damage to target cells T. However, at thisflow rate a typical 7.5 ml fluid sample may pass through the detectorcomponent 12 in 7.5 minutes or less. Similarly, a 3 mL/min flow ratewould indicate a passage of a 7.5 mL sample in about 2.5 minutes.

The target cells and magnetic beads are under the influence of fluidflow that attempts to wash them away from the sensor chip surface aswell as a magnetic force that attempts to draw them to the chip surface.The magnetic force produced by the magnet 32 can thus be calibrated tocounteract the influence of the fluid flow F. In other words, a greaterflow rate may be accomplished by increasing the magnetic force, since agreater force is required to dislodge the cells and beads from the fluidflow. A limiting factor to the strength of the magnetic field generatedby the magnet 32 is that the magnetic force cannot be great enough todisassociate the magnetic beads B from the bound target cells T or greatenough to damage the target cell as the beads are pulled by the magneticforce.

As the fluid sample flows through the first chamber 16 a the magnetattracts the recognition elements M to the plate 18 and lower secondreservoir 16 b. As explained above, most of the unbound beads M_(B) willpass through the openings 51 and into the lower reservoir 16 b wherethey are held in place by the magnetic force. Likewise, the bound targetcells T will be captured against the surface 51 of the apertured plate18 so long as the magnetic force is present. The remaining sample fluid,less the captured target cells, may be delivered to the collectionvessel CC. Alternatively, the valve V5 may be closed and the valve V3opened to allow the sample fluid discharged from outlet 13 b to bereturned to the inlet 13 a via bypass line 43, as reflected in thediagram of FIG. 5C. The use of the bypass can account for any boundtarget cells or any unbound magnetic beads that escape capture withinthe reservoir 16. The sample fluid may be continuously recirculated fora period of time deemed sufficient to capture all of the bound targetcells.

It can be appreciated that at the end of the this second stage of theflow protocol all or at least a majority of the bound target cells T inthe sample fluid have been captured against the surface 51 of theapertured plate 18 within the upper first chamber 16 a. Likewise, all orat least a majority of the unbound magnetic beads R_(B) have been pulledthrough the openings and are collected in the lower second chamber 16 b.The captured target cells are thus available for viewing through theviewing panel 20 in order to count the number of target cells, forinstance. It is contemplated that in a typical procedure the targetcells will be rare or at an extremely low concentration within a sample(e.g., CTCs in a blood sample). Thus, the number of captured cells maybe very low but easily discernible on the apertured plate. In oneapproach the captured cells may be viewed by bright-field microscopy. Inaddition, the captured cells may be further labeled with fluorescentreporters and visualized using fluorescent microscopy. Alternatively orin addition, the magnetic beads may be functionalized with a visualindicator, such as with fluorescent labeling. The magnetic beads may bevisualized using fluorescence microscopy. Since the target cells aretypically bound to a number of magnetic beads the fluorescent image ofthe beads will reveal the presence of the bound target cells. An exampleof captured target cells is shown in the bright-field microscopy imagein FIG. 6. In this image the captured cells are clearly visible. Thecells are MCF-7 (breast cancer cells) that are bound to magnetic beadsfunctionalized with anti-EpCAM antibodies in a known manner. It can benoted that while the great majority of the several million unboundmagnetic beads in the sample passed through the plate openings, someunbound magnetic beads are also present on the plate surface. However,it is apparent that the presence of these few beads does not interferewith a clear view of the captured target cells. In the specific exampleshown in FIG. 6 the openings have a diameter (or smallest effectivedimension) of 5 μm and the magnetic beads have a diameter of 300 nm.

In order to improve visualization of the collected cells, the reservoirmay be washed to eliminate the sample fluid and other cells that mightvisually interfere. In this instance, the magnetic field is maintainedwhile the buffered solution from source B is flowed through the upperand lower portions of the reservoir. The washing cycle may be conductedin the same manner as the initially preparation cycle described above,namely by opening the two chambers 16 a, 16 b to the PBS solution,closing the valve V5 and opening the valve V4 to the buffer collectionvessel BC. Since the magnet remains in position during this washingcycle the target cells will remain captured on the apertured platewithin upper chamber 16 a and the unbound magnetic beads will remaincollected within the lower chamber 16 b.

The microfluidic system 10 disclosed herein is also capable ofcollecting the captured target cells T as well as recovering themagnetic beads M. In one approach, the buffered solution (PBS) is flowedonly through the upper first chamber 16 a with the magnetic fieldremoved. Thus, as shown in the diagram of FIG. 5D, the valve V1 iscontrolled to close flow to the inlet 14 a but open to conduit 35 andvalve V1. Valve V1 is controlled to prevent flow from the source S butaccept the PBS flow from source B. Valves V3 and V4 are closed but valveV5 is open to the collection vessel CC. In the absence of the magneticfield the bound target cells are easily dislodged from the aperturedplate. The flow of PBS can wash the target cells through the outletconduit 23 and into a collection vessel CC. Since the second inlet 14 aand outlet 14 b are closed there is no fluid flow through the lowersecond chamber 16 b. Thus, the collected beads M remain pooled at thebottom of the reservoir 16 even as the target cells are washed away.Alternatively, the magnetic field may be adjusted to reduce the magneticforce experienced by the bound target cells to a level sufficient to beovercome by fluid pressure from the PBS flowing through the upperchamber 16 a. Since the pooled unbound beads in the lower chamber arecloser to the magnet, the magnetic force is sufficient to hold the beadsin place. Once the target cells have been removed and collected themagnetic field can be removed and valves V1 and V4 opened to flow PBSthrough the lower chamber to wash the unbound beads into the collectionvessel BC. Alternatively, the valve V4 can be activated to open thebypass line 44 to redirect the unbound beads back to the sample sourceS. In this instance the unbound beads may be incubated to bind with anypreviously unbound target cells in the original source S or additionalsources Si.

As previously described, the microfluidic system 10 may includeadditional detector components 12 i that may be brought on line by viathe valve V5. In certain protocols is may be contemplated that thesample fluid will flow continuously from the first detector component 12to each successive detector component 12 i before flowing to thecollection vessel CC.

It is contemplated that the conduits and valves be formed of chemicallyinert materials. In a specific example the conduits 35, 36, 38, and 39,and the bypass lines 42-44 may be tubing such as 1/16 inch Cole-Parmertype tubes or other chemically-inert tubing. In certain procedures thesource of target cells may be a conventional 7.5 mL whole blood samplein which the targeted cells have already been bound to recognitionelements, such as magnetic beads. For a typical flow protocol, thebuffer source B may be a 10 mL PBS reservoir or larger. In certainprocedures the sample may be a processed blood sample in which the redblood cells have already been removed by means of lysing or by means ofcommercially available tubes, such as BD Vacutainer Cell PreparationTubes. In other procedures the sample may be other bodily fluids such asurine, or may be water or other fluid samples collected fromenvironmental or industrial sources.

In one embodiment the top and the bottom halves 15 a, 15 b of thedetector component body 15 are machined out of acrylic and fastened byscrews in a manner that sandwiches the apertured plate 18 and seal ring29 to form a fluid tight seal between the chambers. However, othermanufacturing and material are also envisioned, including but notlimited to molded plastic formed in a plastic molding operation.

In one embodiment, the sensor chip apertured plate 18 is about a 15 mmby 15 mm silicon-on-insulator (SOI) wafer having a thickness of about0.5 mm. The openings 50 may be limited to a predetermined active area ofthe detector component of about 10 mm by 10 mm. The array of openings(such as checker-board arrangement) may be defined on the wafer usinglithography and then the holes formed by reactive ion etching of thefront side of the wafer. Individual sensor chips may be defined byreactive ion etching of the back side of the wafer followed by HFetching of the insulating oxide. Alternatively, grooves may be definedusing lithography and etched into the front side of a silicon wafer byreactive ion etching followed by coating with a thin layer of nitride.The nitride on the backside of the wafer can be patterned usinglithography etched to define individual chips. Finally, the siliconwafer can be etched in the opening array pattern using reactive ionetching or potassium hydroxide, and the remaining nitride layer can beremoved by etching. As discussed above, the openings have a smallesteffective dimension that is sufficiently small to trap target cell-boundmagnetic beads, yet sufficiently large to allow free magnetic beads thatare not bound to the target cells to pass therethrough. In a specificexample, the target cells are CTCs, so the openings need to be smallerthan the targeted CTCs but larger than the beads. For example, theaverage size of a lymph node carcinoma of the prostate cells (LNCaP) orovarian cancer cells (IGROV) is about 20 μm while the size of a certaintype of magnetic bead may be about 1 μm. Hence, 3 μm openings will belarge enough to easily pass a free bead but too small to let a CTCthrough. In a specific embodiment, the openings may be provided at about30% packing density, which can result in about 14 openings underneath a20 μm cell. Furthermore, if each cell is bound by multiple magneticbeads (as depicted in FIG. 4), each bead is pulled by the magnetic forceso that the target cell is pulled down in multiple locations, making iteven more difficult for a cell to pass through a single opening. Theopenings are also configured to trap the cell-bound magnetic beads to“lock” the target cells from moving horizontally, preventing them frombeing washed away from the surface of the plate by the fluid flow. Inthe embodiment illustrated in FIGS. 3-4, the openings 50 are shown ashaving a circular or cylindrical with a diameter. However, the openingsmay have other shapes, such as a conical bore or an oblong opening inthe direction of the fluid flow F provided that the smallest effectivedimension of the opening meets the dimensional requirements set forthabove. In an alternative embodiment, the plate 18 may be configured withmicro-grooves each having a width equal to the smallest effectivedimension d discussed above sized so that the target cells cannot enterthe micro-grooves but the much smaller magnetic beads can.

The apertured plate 18 may be coated or passivated with aphysiologically inert material, such as bovine serum albumin (BSA) orpoly ethylene glycol (PEG). Since the system according to the presentdisclosure does not utilize chemical binding between a functionalizedtarget cell and the plate, the surface of the plate can be, and ispreferably, non-reactive.

In accordance with the present disclosure, the target cell-to-magneticbead binding is the only aimed affinity binding step, since the detectorcomponent 12 does not rely on chemically binding the target cells to aportion of the component. Magnetic beads are functionalized in manyconventional ways, including with appropriate monoclonal or polyclonalantibodies (including but not limited to EpCAM antibodies), aptamers orshort peptides that can bind to specific target cells. In an alternativefunctionalization strategy, low molecular weight ligands (e.g. 2-[3-(1,3-dicarboxy propyl)-ureido] pentanedioic acid or “DUPA” for prostatecancer cells, and folic acid for ovarian cancer cells or other cancercells that over-express the folate receptor on their surfaces includinglung, colon, renal and breast cancers) are used to promote binding tocertain cells, most particularly CTCs. Specifically, low molecularweight ligands (e.g. DUPA and folate) can be produced with a functionalgroup (amino, n-hydroxy succinamide (NETS), or biotin depending on thefunctional group on the magnetic bead to be used) with a PEG chain inbetween the low molecular weight ligand and the functional group tosuppress nonspecific binding to the beads.

Functionalized beads are available from a variety of vendors withchemically reactive groups. Magnetic beads are also available in a widerange of sizes (from 100 nm to 5 μm, for example) that can be selectedbased on the dimensions of the target cell to which the beads are bound.In one embodiment NETS-coated 1 μm beads can be the starting point fromChemagen. For these beads, the PEG chain will be terminated with anamino group for covalent linkage to the NETS group on the bead. Thebeads can also be tested from other vendors with other functionalgroups, and can be terminated with the PEG chain and functional groupaccordingly. The beads can also be functionalized with fluorescentmolecules using the appropriate chemistry for the functional group. Forthe example of a low molecular weight ligand, DUPA-PEG-amine andfolate-PEG-amine molecules can be synthesized which can be reacted withthe beads before fluorescent labeling. Thereafter, the desired amount ofreactive fluorescent dye (fluorescein, for example) can be reacted withthe beads, after which residual NHS groups (or other activated moietieson the beads) can be passivated by reaction with glucosamine (or anotherappropriate molecule for neutralizing the activated moiety on thebeads). The ratio of folate-PEG-amine or DUPA-PEG-amine to fluorescentdye and passivating molecule can be optimized, as needed. Similarly,antibodies (such as the epithelial cell adhesion molecule or “EpCAM”)can be immobilized on beads. One commercially available example, inwhich the functionalized magnetic beads are magnetic beadsfunctionalized by attachment to a monoclonal antibody against the humanEpithelial Cell Adhesion Molecule (EpCAM), such as Dynabeads® EpithelialEnrich, commercially available from Invitrogen. For example antibodiescan be covalently linked to NETS-coated beads, or can be linked tostreptavidin-coated beads via a biotin. Various other functionalizationschemes can also be used including but not limited to carboxyl groups,thiols, and silanes. Alternatively, the beads may only have therecognition elements to bind and trap the cells on the plate surface butlack the fluorescent reporters which could be introduced separately tobind directly to the captured cells. In one procedure, fluorescentlylabeled antibodies (e.g. cytokeratin), low molecular weight ligands,peptides or aptamers can be separately exposed to the captured cells.

The microfluidic system 10 and detector component 12 disclosed hereinare particularly suited to detection of cancer cells bound to magneticbeads. Many techniques are available for functionalizing and bindingmagnetic beads to target cells such as CTCs. Exemplary procedures aredisclosed in the published the following publications: T. Mitrelias, etal., “Biological cell detection using ferromagnetic microbeads,” Journalof Magnetism and Magnetic Materials, vol. 310, pp. 2862-2864, March2007; N. Eide, et al., “Immunomagnetic detection of micrometastaticcells in bone marrow in uveal melanoma patients,” Acta Ophthalmologica,vol. 87, pp. 830-836, December 2009; Yu et al., “Circulating tumorcells: approaches to isolation and characterization”, The Journal ofCell Biology, Vol. 192, No. 3, pp. 373-382 (Feb. 7, 2011); Hayes &Smerage, “Circulating Tumor Cells”, Progress in Molecular Biology andTranslational Science, Vol. 95, 2010, pp. 95-112; Alexiou et al.,“Medical Applications of Magnetic Nanoparticles”, Journal of Nanoscienceand Nanotechnology, Vol. 6, 2006, pp. 2762-2768; and Ito, et al.,“Medical application of functionalized magnetic nanoparticles”, Journalof Bioscience and Bioengineering, Vol. 100, 2005, pp. 1-11, thedisclosure of each publication being incorporated herein by reference.

Publications disclosing exemplary procedures for synthesis andmodification of folate and DUPA are identified in the Appendix. Any ofthe procedures and methods disclosed in these publications may besuitable for binding magnetic beads to select CTCs that may besubsequently captured by the detector component 12 disclosed herein.Functionalization of magnetic beads with folate is fully described inreferences listed in the Appendix, therefore one having ordinary skillin the art is enabled to functionalize similar magnetic beads with DUPAas further described in the listed references. The DUPA molecule itselfis also fully described in references listed in the Appendix andelsewhere in this specification.

The ability to attract bead-bound cancer cells to a solid surface (i.e.,without any openings) has been verified in experiments using MCF-7 cells(breast cancer cell line) attached to magnetic beads via EpCAMantibodies. The target cells were flowed with volumetric flow rateshigher than 2 mL/minute and were successfully magnetically attracted tothe solid surface during the flow. The target cells have a smallesteffective dimension of about 20 μm so in another experiment the targetcells were flowed over a plate having openings with an effectivediameter of 5 μm. Intact cancer cells were observed on the plate usingdual surface (cytokeratin) and nuclear (DAPI) staining. In theseexperiments 9 out of 10 target cells were detected in a 12 mL sample,for a 90% cell recovery.

The operation microfluidic detection system 10 disclosed herein can becontrolled through a master controller 45. The controller may be amicroprocessor configured to follow a controlled flow protocol accordingto a particular target cell, recognition element and sample size. Themaster controller may incorporate a reader to read indicia associatedwith a particular sample or samples, and automatically upload andexecute a predetermined flow protocol associated with the particularsample.

The controller 45 may also be configured to allow user-controlledoperation. For instance, the flow rate for a particular targetcell-magnetic bead combination can be optimized by increasing the flowrate of a bound target cell sample until it is no longer possible toattract beads to the surface 51 of the apertured plate 18. Thecontinuous operation of the system may be directly observed through thevisualization window to determine whether a flow bypass is required orwhether the detection process is complete.

In the illustrated embodiments the magnetic beads are described may befunctionalized with a fluorescent marker. In these embodiments theapertured plate 18 is generally opaque so fluorescence signals comingfrom the unbound beads M_(B) in the bottom chamber 16 b will not bedetected through the viewing panel 20. However, in cases some free beadsmay remain on the surface 51 and produce fluorescence signals that canconfuse the visualization. In these cases the beads can befunctionalized only with ligands and not with fluorescent markers. Afterthe sample has been fully processed and the target cells captured on theapertured plate 18, fluorescent-tagged ligands may be introducedseparately into the reservoir to bind to the target cells directly. Thetarget cells can then be easily observed through the visualization panel20. With this modified approach, in some cases the plate 18 can bedevoid of any micro-apertures since the target cells on the platesurface can be readily differentiated from unbound magnetic beads bymeans of fluorescence.

In the detection process, the lower chamber 16 b can be flooded with aminute amount of buffer or blood, after which here is no fluid flowthrough the lower chamber until the detection is complete. Fluiddiffusion through the openings 50 between the upper and lower chambers16 a, 16 b will be minimal since this microfluidic detection system 10is not based on a pressure-driven flow.

CTCs from blood samples of patients of various cancers, including butnot limited to prostate, ovarian breast, colon, renal and lung cancers,can be detected since many cancer cells express certain molecules orantigens on their surfaces which can be targeted with variousrecognition elements, including but not limited to antibodies (e.g.,EpCAM), aptamers, low molecular weight ligands (e.g., folate and DUPA)and peptides. The beads can be functionalized as previously described(e.g., DUPA for prostate cancer cells, and folate end EpCAM for ovarian,breast, colon, renal and lung cancer cells) and then incubated and mixedwith the sample fluid for 20-30 minutes. This incubation time may belonger or shorter depending on the number of beads used, sample volumeand the number of target cells sought. For instance, seeding the samplewith a larger number of beads increases the chance that a target cellwill “find” a magnetic bead and bind. If there are multiple samples,incubation of all samples can be carried out simultaneously. Theanalysis of the sample fluid by the present detection system, and thesequence of sample fluid and buffer flows can be carried out asdescribed herein. Aliquots of the captured cancer cells can be stainedwith additional recognition elements, such as antibodies to cytokeratinsand EpCAM to assure that the cells retained by the detector componentare indeed cancer cells. Furthermore, a preliminary indication ofwhether CTC numbers correlate with the stage of the disease of thesample donor can be ascertained. While the disclosed system is notlimited to a particular type of cancer, prostate and ovarian cancercells are especially mentioned herein as models for the system becauseboth diseases can have vague symptoms resulting in confusingbiomolecular tests, and both can benefit from a reliable, fast andsensitive CTC test. For example prostate cancer can have symptomssimilar to benign prostatic hyperplasia. PSA biomarker tests can beconfusing due to both false positives and false negatives. Similarly,ovarian cancer can have vague symptoms and may be detected as late asstage III or IV.

In comparison with a number of studies which successfully used magneticbeads to manually separate a wide variety of cells (from pathogens to Tcells to CTCs) from complex samples, the approach according to thepresent disclosure advantageously offers separation-and-detection,faster analysis and the ability to harvest the captured cells and makethem available for other types of analyses, including but not limited togenetic analysis. Furthermore, in comparison with giant magnetoresistive (GMR) or spin-valve sensors which are known to a person ofordinary skill in the art (made of multiple nanolayers withprecisely-controlled thicknesses) that can detect magnetic beads, thepresent approach advantageously is more robust, easy to construct anduse, and offers much higher throughput. The system according to thepresent disclosure also offers a significant advantage over size-basedcell entrapment assays which force the cells through micron-sizedcavities by fluidic pressure. These assays can suffer from clogging ofthe cavities (since all entities in the sample are forced to passthrough the cavities), or trapping of other entities similar in size totarget cells, or complete passage and hence loss of target cells throughthe cavity.

The source S may include target cells that have already been bound torecognition elements, as well as magnetic beads. Alternatively thesource may initially contain a sample fluid, such as a whole bloodspecimen, to which magnetic beads are added and allowed to incubate.Since the target cells are rare or at a low concentration, it isdesirable to seed the specimen with millions of functionalized magneticbeads. For instance, in one approach 100 million beads are provided foreach mL of whole blood specimen. A twenty minute incubation time hasbeen found to be sufficient to bind rare CTCs. The system 10 may beprimed as described above during the incubation period since the samplesource S is not involved in the fluid flow during this step. Once theincubation period is complete the flow protocol for detecting the targetcells described above may be implemented. Alternatively, as describedabove, the sample provided may be a blood sampled processed by acombination of commercial cell preparation tubes and centrifugation inorder to discard red blood cells which are usually not sought during aCTC detection. Alternatively, the sample may be a processed blood samplewherein the red blood cells have been lysed using a red blood cell lysisbuffer.

It is contemplated that the system 10 may be modified for incorporationinto a dialysis or dialysis-type system. In this instance,functionalized magnetic beads may be injected into the patient's bloodstream prior to dialysis. Rather than flowing into a collection vesselCC the blood flowing through the detector component 12 or detectorcomponents 12 i is returned to the dialysis. A magnetic collectionelement may be incorporated at the system output to capture all unboundmagnetic beads and bound cells that have not been collected within thedetector component(s).

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Therefore,above disclosure is not to be limited to the specific embodimentsillustrated and described above. The description as presented and asthey may be amended, encompass variations, alternatives, modifications,improvements, equivalents, and substantial equivalents of theembodiments and teachings disclosed herein, including those that arepresently unforeseen or unappreciated, and that, for example, may arisefrom applicants/patentees and others.

For instance, in the exemplary embodiments particular biological cells,such as CTCs, are described as being captured by the detector component12 of the microfluidic detection system 10. Other entities, includingparticles or similar bodies, in the micrometer (μm) size range may alsobe detected and captured by the system disclosed herein, provided theparticles or bodies can bind to one or more recognition elements, suchas the magnetic beads described herein.

Furthermore, the apertured plate 18 is depicted as being generallyparallel with the lower half 15 b of the detector component base 15.Alternatively the plate may be angled toward the outlet channel 23 sothat captured target cells tend to accumulate from the outlet end andfill in toward the inlet end. An angled plate may also facilitatepassage of the unbound magnetic beads into the lower chamber 16 b byinducing a translation along the surface 51 of the plate. Moreover, theplate 18 is shown as generally planar, although other configurations arecontemplated that facilitate capturing target cells and passage ofunbound beads.

The magnet 32 is described herein as a permanent magnet with theapplication of the magnetic field controlled by moving the magnet. Themagnetic field may be manipulated by an adjustable shield disposedbetween the magnet and the reservoir 16. The shield may be used tocompletely block the magnetic field or to reduce the field as necessary.Alternatively the magnet may be an electromagnet that can be controlledby the controller 45 to activate or de-active the magnetic field oradjust the strength of the field. The controller may also modulate themagnetic field during a detection cycle to facilitate capturing thetarget cells and drawing the unbound magnetic beads into the lowerchamber.

In a further alternative the magnet 32 may include multiple magnetsarranged in a predetermined pattern to facilitate counting cellscaptured on the apertured plate 18. Thus, in one embodiment severalmagnets may be arranged in parallel strips so that the captured cellsappear in several lines.

In the fluid flow protocols described above, the target cells areflushed from the detector component 12 in one step. Alternatively thecells may be retained on the apertured plate and the plate itselfremoved from the detector component. In this alternative the magneticcan remain in position as the upper body half 15 a is removed to provideaccess to the apertured plate. The lower body half 15 b may betransported with the apertured plate and magnet intact and mated withanother body half for further procedures.

What is claimed is:
 1. A system for isolating target entities from afluid, the system comprising: (a) a plurality of magnetic beads, whereineach of the magnetic beads is capable of binding to a target entity whenthe magnetic beads are added to a fluid containing the target entities,and wherein the magnetic beads are smaller than the target entities; (b)a detector component comprising: a body defining a reservoir; and aplate separating the reservoir into a first chamber and a secondchamber, wherein the plate defines a plurality of openings therethrough,wherein each opening is sized to permit passage of the magnetic beadsand to prevent passage of the target entities, and wherein the bodycomprises a first inlet to the first chamber and a first outlet from thefirst chamber; and (c) a magnet affixed to or supported relative to anouter surface of the detector component and arranged so that the secondchamber and the plate are situated between the magnet and the firstchamber, wherein the magnet generates a magnetic force at a surface ofthe plate facing the first chamber sufficient (i) to attract at leastsome of the magnetic beads not bound to a target entity through theopenings in the plate from the first chamber into the second chamber and(ii) to attract and hold at least some of the target entities bound tothe magnetic beads against the surface of the plate such that the targetentities are not dislodged from the surface when fluid flows through thefirst chamber.
 2. The system of claim 1, wherein the first chamber issufficiently large to enable a flow of fluid through the first chamberat a flow rate of at least 1 milliliter per minute and at a linear flowvelocity that is sufficiently low to avoid damage to the target entitiesand to avoid dislodging the target entities from the surface of theplate in the first chamber.
 3. The system of claim 2, wherein the linearflow velocity is less than about 70 mm/second.
 4. The system of claim 1,wherein the detector component further comprises a viewing panelarranged to enable a view of the surface of the plate facing the firstchamber.
 5. The system of claim 4, wherein the viewing panel is anoptically transparent glass.
 6. The system of claim 1, wherein the bodyfurther comprises a first inlet channel extending from the first inletto the first chamber.
 7. The system of claim 1, wherein the body furthercomprises a cavity on the outer surface of the detector componentarranged to accommodate the magnet.
 8. The system of claim 1, whereinthe body further comprises a second inlet to the second chamber and asecond outlet from the second chamber.
 9. The system of claim 1, furthercomprising a pump and first and second conduits, wherein the pumpcomprises a pump inlet connected to the first outlet by the firstconduit, and a pump outlet connected by the second conduit to acollection vessel for receiving fluid pumped through the first chamberand out through the first outlet.
 10. The system of claim 9, furthercomprising: a first controllable valve disposed between the first outletand the second outlet and the pump inlet; and a separate secondcontrollable valve disposed downstream of the pump outlet.
 11. Thesystem of claim 9, further comprising: a bypass conduit connected to thesecond outlet and fluidly connectable to a source of the fluidcontaining the target entities; and a controllable valve disposedbetween the second conduit and the bypass conduit for selectivelydirecting fluid pumped through the second outlet to either the bypassconduit or a non-reactive fluid collection vessel.
 12. The system ofclaim 9, further comprising: a bypass conduit arranged between thesecond conduit and the first inlet; and a controllable valve fordirecting fluid flowing through the first outlet to either thecollection vessel or the bypass conduit.
 13. The system of claim 1,wherein the surface of the plate is provided with a physiologicallyinert coating.
 14. The system of claim 1, wherein the magnetic beadscomprise a recognition element that is capable of binding specificallyto a target entity.
 15. The system of claim 1, wherein each magneticbead has a largest effective dimension of about 100 nm to about 5.0microns.
 16. The system of claim 1, wherein each of the openings in theplate has an effective dimension of about 3.0 to about 5.0 microns. 17.A method for isolating target entities from a fluid, the methodcomprising: obtaining a fluid that may contain target entities; mixingthe fluid with a plurality of magnetic beads to form a fluid mixture,wherein each of the magnetic beads is capable of binding to a targetentity when the magnetic beads are added to the fluid containing thetarget entities, and wherein the magnetic beads are smaller than thetarget entities; flowing the fluid mixture through a detector component,wherein the detector component comprises a body defining a reservoir;and a plate separating the reservoir into a first chamber and a secondchamber, wherein the plate defines a plurality of openings therethrough,wherein each opening is sized to permit passage of the magnetic beadsand to prevent passage of the target entities; and applying a firstmagnetic force at a surface of the plate facing the first chambersufficient (i) to attract at least some of the magnetic beads not boundto a target entity through the openings in the plate from the firstchamber into the second chamber and (ii) to attract and hold at leastsome of the target entities bound to the magnetic beads against thesurface of the plate such that the target entities are not dislodgedfrom the surface by the fluid flowing through the first chamber.
 18. Themethod of claim 17, wherein the fluid is a bodily fluid and the targetentities are rare cells comprising circulating tumor cells or fetalcells in maternal blood.
 19. The method of claim 18, wherein the rarecells are the circulating tumor cells (CTCs).
 20. The method of claim18, wherein the rare cells and/or the magnetic beads have beenfunctionalized with a fluorescent marker.
 21. The method of claim 18,wherein the rare cells are the fetal cells in the maternal blood.